EP2925884B1 - Zusammensetzungen und verfahren zur bewertung von herzfehlern - Google Patents

Zusammensetzungen und verfahren zur bewertung von herzfehlern Download PDF

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EP2925884B1
EP2925884B1 EP13802567.1A EP13802567A EP2925884B1 EP 2925884 B1 EP2925884 B1 EP 2925884B1 EP 13802567 A EP13802567 A EP 13802567A EP 2925884 B1 EP2925884 B1 EP 2925884B1
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patient
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bnp
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EP2925884A1 (de
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Yvan Devaux
Mélanie VAUSORT
Lu Zhang
Daniel Wagner
Iain SQUIRE
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Luxembourg Institute of Health LIH
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/141—MicroRNAs, miRNAs
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to compositions and kits comprising probes for detecting miRNAs useful for monitoring the diagnosis or progression of heart disease in an individual.
  • the compositions of the invention can be used for the prognosis of patients having suffered from an acute myocardial infarction.
  • Heart disease encompasses a family of disorders, such as cardiomyopathies, and is a leading cause of morbidity and mortality in the industrialized world. Disorders within the heart disease spectrum are understood to arise from pathogenic changes in distinct cell types, such as cardiomyocytes, via alterations in a complex set of biochemical pathways.
  • Left ventricular (LV) remodelling develops after acute myocardial infarction (AMI) in a significant proportion of patients 1 . Associated mortality and morbidity are important and may be prevented or at least alleviated by personalized health care. To achieve this goal, however, it is critical to identify new tools to accurately predict the development of LV remodelling.
  • N-terminal pro-brain natriuretic peptide (Nt-pro-BNP) is known to be associated with LV dysfunction after AMI, it fluctuates after AMI and better predicts poor outcome when measured 3-5 days after AMI 2 .
  • Talwar S et al (2000) Eur. Heart J. 21:1514-1521 showed that Nt-pro-BNP was an independent predictor of wall motion index score (WMIS), an indicator of LV contractility and remodelling.
  • WMIS wall motion index score
  • miRNAs microRNAs
  • Their potential to diagnose AMI has been suggested by multiple reports 5 , 6 .
  • their prognostic value has received much less attention, and only cardiomyocytes-enriched miRNAs have been evaluated 7 and WO2008042231 .
  • the temporal profile of circulating miRNAs is related to the development of LV remodelling after AMI 8 , which suggests their usefulness as prognostic biomarkers.
  • WO 2008/043521 discloses a large number of miRNAs, including those of the present invention, for evaluating and treating a cardiac disease.
  • WO 2008/042231 discloses a list of microRNAs, including miR-101 and miR-27a, as suitable markers for evaluating heart diseases.
  • a group of 4 miRNAs miR-16 encoded by SEQ ID NO:1, miR-27a encoded by SEQ ID NO:2, miR-101, encoded by SEQ ID NO:3, miR-150 encoded by SEQ ID NO:4, (i.e. further indicated as the miRNA panel of the invention) can add to the predictive value (or prognostic value) of the existing marker, i.e. Nt-pro-BNP, in a prospective cohort of AMI patients.
  • the potential of the miRNA panel was shown to aid in the prognostication of patients having suffered from acute myocardial infarction.
  • the four miRNAs of the present invention were selected from a pool of 695 possible miRNAs and, surprisingly, it has been found that only these four, in specific combination, are able to enhance the prognosis of left ventricular remodelling, preferably in combination with Nt-pro-BNP.
  • the present invention shows an added value of the 4 miRNA panel to Nt-pro-BNP as shown in SEQ ID NO:5, to classify patients which have suffered from myocardial infarction.
  • the sensitivity of the prediction was improved, and the specificity was preserved.
  • the invention provides a kit for monitoring the prognosis of a patient having suffered from acute myocardial ischemia consisting of (1) probes for measuring a panel of miRNA biomarkers in a sample of bodily fluid of said patient, said panel of miRNA biomarkers consisting of: (a) miR-16 encoded by SEQ ID NO:1, (b) miR-27a encoded by SEQ ID NO:2, (c) miR-101 encoded by SEQ ID NO:3, and (d) miR-150 encoded by SEQ ID NO:4, and optionally (2) detection reagents for measuring Nt-pro-BNP as shown in SEQ ID NO:5 in a sample of bodily fluid of said patient.
  • said panel of miRNA biomarkers consisting of: (
  • the invention provides said kit for detecting said biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • the invention provides the kit for detecting said biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 and Nt-pro-BNP for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • the invention provides the use of a kit for detecting a biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • the invention provides the use of a kit for detecting said biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 and Nt-pro-BNP for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • the invention provides a method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction comprising determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient and correlating the levels of said miRNAs with a previously established classification model wherein said model was developed by fitting data from a study of a population of patients and said fitted data comprises levels of said biomarkers and conversion to the development of left ventricular remodeling in said selected population of patients and optionally also determining an increase in levels of Nt-pro-BNP by comparison with the control and wherein a prognostic score is obtained for being at risk of developing left ventricular modeling by establishing the odds ratios of only said miRNAs optionally in combination with levels of Nt-pro-BNP.
  • the invention provides a method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction comprising determining the levels of miR-16, miR-27a, miR-101, miR-150 and Nt-pro-BNP in a body fluid of said patient and correlating the levels of said miRNAs and Nt-pro-BNP with a previously established classification model wherein said model was developed by fitting data from a study of a population of patients and said fitted data comprises levels of said biomarkers and conversion to the development of left ventricular remodeling in said selected population of patients and optionally also determining an increase in levels of Nt-pro-BNP by comparison with the control and wherein a prognostic score is obtained for being at risk of developing left ventricular modeling by establishing the odds ratios of only said miRNAs optionally in combination with levels of Nt-pro-BNP.
  • the patient having suffered from an acute myocardial infarction has a WMIS score between 1 and 1.4.
  • the invention provides a method for assessing the efficacy of a treatment for a patient having suffered from an acute myocardial infarction and having a likelihood of developing a reduced LV contractility wherein the method comprises i) determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient, ii) determining the Nt-pro-BNP level in a body fluid of said patient, iii) determining the levels of miR-16, miR-27a, miR-101 and miR-150 and the level of Nt-pro-BNP in a body fluid of said patient after treatment, iv) comparing the results of i) and ii) with the results of iii), wherein a difference between the results of i), ii) and iii) indicates an effect of the treatment.
  • a patient has a WMIS score between 1 and 1.4.
  • the body fluid is blood, plasma or serum.
  • the application further discloses a composition of i) at least one short interfering nucleic acid capable of encoding a miRNA selected from the list consisting of miR-101 and miR-150 and at least one short interfering nucleic acid capable of inhibiting a miRNA selected from the list consisting of miR-16 and miR-27a or ii) short interfering nucleic acids capable of encoding miR-101 and miR-150 or iii) short interfering nucleic acids capable of inhibiting miR-16 and miR-27a for the treatment of left ventricular remodeling.
  • the application further discloses pharmaceutical formulations comprising the previous compositions.
  • the prognostic value of an assay comprising a panel of 4 different miRNAs in AMI patients.
  • a panel of 4 specific miRNAs i.e. miR-16, miR-27a, miR-101 and miR-150
  • the determination of Nt-pro-BNP is found to improve the prognostic value of the gold standard Nt-pro-BNP as a stand-alone prognostic marker.
  • the method of the invention increases the sensitivity from 48 to 60%, while maintaining the specificity at 75%.
  • the positive predictive value was increased form 67% to 71%, and the negative predictive value was increased form 58% to 64%.
  • One particular advantage of the invention is that the method for prognosis also improves the classification of patients with intermediate phenotypes, particularly dyskinetic patients, which are difficult to classify using existing biomarkers.
  • the application discloses a biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 and the invention provides in a first embodiment a kit comprising the probes detecting said panel for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • a biomarker panel consisting of miR-16, miR-27a, miR-101, miR-150 and Nt-pro-BNP and a further embodiment of the invention provides a kit comprising probes and/or reagents detecting said panel for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • kits for detecting a biomarker panel consisting of miR-16, miR-27a, miR-101 and miR-150 for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • kits for detecting a biomarker panel consisting of miR-16, miR-27a, miR-101, miR-150 and Nt-pro-BNP for monitoring the prognosis of a patient having suffered from acute myocardial ischemia.
  • the invention provides a method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction comprising determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient and correlating the levels of said miRNAs with a previously established classification model wherein said model was developed by fitting data from a study of a population of patients and said fitted data comprises levels of said biomarkers and conversion to the development of left ventricular remodeling in said selected population of patients, and optionally also determining an increase in levels of Nt-pro-BNP by comparison with the control, and wherein a prognostic score is obtained for being at risk of developing left ventricular modeling by establishing the odds ratios of only said miRNAs optionally in combination with levels of Nt-pro-BNP.
  • the invention provides a method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction comprising determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient and correlating the levels of said miRNAs with a previously established classification model wherein said model was developed by fitting data from a study of a population of patients and said fitted data comprises levels of said biomarkers and conversion to the development of left ventricular remodeling in said selected population of patients, and optionally also determining an increase in levels of Nt-pro-BNP by comparison with the control, and wherein a prognostic score is obtained for being at risk of developing left ventricular modeling by establishing the odds ratios of only said miRNAs optionally in combination with levels of Nt-pro-BNP and wherein a practitioner may start a treatment plan based on the prognostic score.
  • the treatment plan involves the administration of a drug, such as an ACE inhibitor, an angiotensin II receptor blocker, a Beta-blocker, a vasodilator, a pro-angiogenic factor, a cardiac glycoside, an antiarrhythmic agent, a diuretic, a statin, or an anticoagulant, an inotropic agent; an immunosuppressive agent, use of a pacemaker, defibrillator, mechanical circulatory support, surgery, or therapy with stem cells (bone marrow derived stem cells, mesenchymal stem cells, cardiac stem cells, muscle derived stem cells).
  • a drug such as an ACE inhibitor, an angiotensin II receptor blocker, a Beta-blocker, a vasodilator, a pro-angiogenic factor, a cardiac glycoside, an antiarrhythmic agent, a diuretic, a statin, or an anticoagulant, an inotropic agent
  • an immunosuppressive agent use of a pacemaker, defibrill
  • the invention provides a method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction comprising: i) determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient, ii) determining the Nt-pro-BNP level in a body fluid of said patient wherein the levels of said miRNAs and said Nt-pro-BNP level is correlated with a previously established classification model wherein said model was developed by fitting data from a study of a population of patients and said fitted data comprises levels of said biomarkers and conversion to the development of left ventricular remodeling in said selected population of patients and wherein a prognostic score is obtained for being at risk of developing left ventricular modeling.
  • the body fluid for measuring the levels of Nt-pro-BNP and the body fluid for measuring the levels of miR-16, miR-27a, miR-101 and miR-150 are different body fluids.
  • a body fluid is blood, serum, plasma, Cerebro Spinal Fluid (CSF), saliva or urine.
  • CSF Cerebro Spinal Fluid
  • the body fluid is blood, serum or plasma.
  • the body fluid of a patient having suffered from an acute myocardial infarction is sampled after 5 minutes, 10 minutes, 60 minutes, 2 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days or after even a longer period.
  • the body fluid is sampled at any time point between 5 minutes and 4 weeks after the acute myocardial infarction.
  • Plasma and serum preparation are well known in the art. Either "fresh" blood plasma or serum, or frozen (stored) and subsequently thawed plasma or serum may be used. Frozen (stored) plasma or serum should optimally be maintained at storage conditions of -20 to -70°C until thawed and used. "Fresh” plasma or serum should be refrigerated or maintained on ice until used, with nucleic acid extraction being performed as soon as possible. Blood can be drawn by standard methods into a collection tube, typically siliconized glass, either without anticoagulant for preparation of serum, or with EDTA, sodium citrate, heparin, or similar anticoagulants for preparation of plasma.
  • plasma or serum When preparing plasma or serum for storage, although not an absolute requirement, is that plasma or serum is first fractionated from whole blood prior to being frozen. This reduces the burden of extraneous intracellular RNA released from lysis of frozen and thawed cells which might reduce the sensitivity of the amplification assay or interfere with the amplification assay through release of inhibitors to PCR such as porphyrins and hematin.
  • "Fresh" plasma or serum may be fractionated from whole blood by centrifugation, using gentle centrifugation at 300-800 times gravity for five to ten minutes, or fractionated by other standard methods. High centrifugation rates capable of fractionating out apoptotic bodies should be avoided.
  • An AMI patient is a patient who has suffered from an acute myocardial infarction.
  • the classification model is established with patients who have suffered from an acute myocardial infarction.
  • patients are recruited who developed left ventricular remodeling and patients who did not develop left ventricular remodeling.
  • a method for predicting and/or monitoring the prognosis refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient.
  • the term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given characteristic, such as the presence or level of a prognostic indicator, when compared to those individuals not exhibiting the characteristic.
  • an AMI patient exhibiting a high level of miR-16 and mi-R27a and a low level of miR-150 and miR-101 and an increased level of Nt-pro-BNP, as compared to a mean value determined in a population of patients included in the classification model, may be more likely to suffer or to progress towards a patient with an impaired LV contractility.
  • a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance.
  • the term "about” in this context refers to +/- 1%.
  • associating a prognostic indicator with a predisposition to an outcome of reduced LV contractility is a statistical analysis.
  • changes in the miRNA panel as described herein in combination with a change in the amount of Nt-pro-BNP may signal that a patient, in particular an AMI patient, is more likely to suffer from an adverse outcome than patients with different levels, as determined by a level of statistical significance.
  • Common tests for evaluating statistical significance include but are not limited to ANOVA, Kniskal-Wallis, t-test and odds ratio (OR).
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval (CI) and/or a p value.
  • Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.
  • correlating refers to comparing the presence or amount of the prognostic indicator in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition.
  • the miRNA panel levels in a patient can be compared to a level known to be associated with an increased disposition of developing an impaired left ventricular contractility.
  • the patient's miRNA panel levels are said to have been correlated with a prognosis; that is, the skilled artisan can use the patient's miRNA panel levels, optionally in combination with the determination of the Nt-pro-BNP levels, to determine the likelihood that the patient is at risk for developing impaired LV contractility or dyskinesia, and respond accordingly.
  • the patient's miRNA panel levels can be compared to a miRNA panel level known to be associated with a good outcome (e.g., no impaired LV contractility, no risk for sudden death, etc.), and determine if the patient's prognosis is predisposed to the good outcome.
  • expression pattern refers to the combination of occurrences or levels in a set of miRNAs of a sample.
  • a comparison is made between the occurrences or levels of the same miRNAs in the test and reference (or control) expression patterns for each of the four miRNA pairs.
  • the classification scheme involves building or constructing a statistical model also referred to as a classifier or predictor, that can be used to classify samples to be tested (test samples) based on miRNA levels or occurrences.
  • the model is built using reference samples (control samples) for which the classification has already been ascertained, referred to herein as a reference dataset comprising reference expression patterns.
  • reference expression patterns are levels or occurrences of a set of one or more miRNAs in a reference sample (e.g. a reference blood or plasma or serum sample).
  • a test expression pattern obtained from a test sample is evaluated against the model (e.g. classified as a function of relative miRNAs expression of the sample with respect to that of the model).
  • evaluation involves identifying the reference expression pattern that most closely resembles the expression pattern of the test sample and associating the known reduced left ventricular contractility class or type of the reference expression pattern with the test expression pattern, thereby classify (categorizing) the risk towards developing a reduced left ventricular contractility associated with the test expression pattern.
  • the number of relevant miRNAs to be used for building the model can be determined by one of skill in the art.
  • a greedy search method backward selection
  • Support Vector Machine is used to determine a subset of miRNAs that can be chosen to build a model (e.g., Naive Bayes and Logistic regression) for prediction of the presence of left ventricular contractility reduction.
  • a class prediction strength can also be measured to determine the degree of confidence with which the model classifies a sample to be tested. The prediction strength conveys the degree of confidence of the classification of the sample and evaluates when a sample cannot be classified. There may be instances in which a sample is tested, but does not belong to a particular class.
  • a threshold wherein a sample which scores below the determined threshold is not a sample that can be classified (e.g., a "no call”).
  • the prediction strength threshold can be determined by the skilled artisan based on known factors, including, but not limited to the value of a false positive classification versus a "no call.”
  • This process is done with all the samples of the initial dataset and an error rate is determined. The accuracy of the model is then assessed.
  • This model classifies samples to be tested with high accuracy for classes that are known, or classes that have been previously ascertained or established through class discovery as discussed herein.
  • Another way to validate the model is to apply the model to an independent data set, such as a new unknown test plasma or blood or serum sample. Other standard biological or medical research techniques, known or developed in the future, can be used to validate class discovery or class prediction.
  • Classification of the sample gives a healthcare provider information about a classification to which the sample belongs, based on the analysis of the levels of the miRNA panel of the invention, optionally including the determination of Nt-pro-BNP levels.
  • the information provided by the present invention aids the healthcare provider in diagnosing the individual.
  • the present invention provides methods for determining a treatment plan. Once the health care provider knows to which disease class (i.e. being at risk for developing LV remodeling or not) the sample, and therefore, the individual belongs, the health care provider can determine an adequate treatment plan for the individual. For example, different assessments of left ventricular contractility reduction often require differing treatments.
  • Properly diagnosing and understanding the seriousness of left ventricular remodeling of an individual allows for a better, more successful treatment and prognosis.
  • Other applications of the invention include classifying persons who are likely to have successful treatment with a particular drug or therapeutic regiment. Those interested in determining the efficacy of a drug for reducing left ventricular remodeling can utilize the methods of the present invention.
  • the invention relates to a method of assessing the efficacy of a treatment for a patient having suffered from an acute myocardial infarction and is at risk for developing a reduced LV contractility wherein the method comprises i) determining the levels of miR-16, miR-27a, miR-101 and miR-150 in a body fluid of said patient, ii) determining the Nt-pro-BNP level in a body fluid of said patient, iii) determining the levels of miR-16, miR-27a, miR-101 and miR-150 and the level of Nt-pro-BNP in a body fluid of said patient after treatment, iv) comparing the results of i) and ii) with the results of iii), wherein a difference between the results of i), ii) and iii) indicates an effect of the treatment.
  • the treatment is the administration of a drug, such as an ACE inhibitor, an angiotensin II receptor blocker, a Beta-blocker, a vasodilator, a cardiac glycoside, an antiarrhythmic agent, a diuretic, statins, or an anticoagulant, an inotropic agent; an immunosuppressive agent, use of a pacemaker, defibrillator, mechanical circulatory support, or surgery.
  • a drug such as an ACE inhibitor, an angiotensin II receptor blocker, a Beta-blocker, a vasodilator, a cardiac glycoside, an antiarrhythmic agent, a diuretic, statins, or an anticoagulant, an inotropic agent; an immunosuppressive agent, use of a pacemaker, defibrillator, mechanical circulatory support, or surgery.
  • Assay measurement strategies numerous methods and devices are well known to the skilled artisan for measuring the prognostic indicators of the instant invention. With regard to polypeptides, such as Nt-pro-BNP, in patient samples, immunoassay devices and methods are often used. See, e.g. US6143576 , US6113855 and US6019944 . These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g. US5631171 and US5955377 .
  • the expression of these 4 miRNAs can be measured separately or simultaneously.
  • the miRNA expression levels are obtained, e.g. by using a quantitative RT-PCR or a bead-based system.
  • a suitable array-based system e.g. miRMAX microarray, GeneXpert System Cepheid, MDx platform Biocartis
  • miRMAX microarray GeneXpert System Cepheid, MDx platform Biocartis
  • the levels of miR-150 and miR101 in the body fluid derived from a patient having suffered from an AMI and being at risk for developing an impaired LV contractility are lower than the corresponding miRNA levels in the corresponding body fluid of a group of control patients.
  • a control patient is typically a patient having suffered from an AMI and having preserved LV contractility.
  • a control patient is a patient who has not suffered from an AMI and is also an individual with a preserved LV contractility.
  • the levels of miR-150 and miR101 in the body fluid derived from a patient having suffered from an AMI and being at risk for developing an impaired LV contractility are at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower than the levels of the corresponding miRNA levels in the corresponding body fluid of a control patient.
  • the levels of miR-16 and miR27a in the body fluid derived from a patient having suffered from an AMI and being at risk for developing an impaired LV contractility are higher than the corresponding miRNA levels in the corresponding body fluid of a control patient.
  • the levels of miR-16 and miR27a in the body fluid derived from a patient having suffered from an AMI and being at risk for developing an impaired LV contractility are at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher than the levels of the corresponding miRNA levels in the corresponding body fluid of a control patient.
  • the likelihood of classifying said patient into the category (or class) of developing a reduced left ventricular contractility is increased by 4.2 fold and for each increase of 1 unit of miR-27a in the patient body fluid the likelihood of classifying said patient into the category of developing a reduced left ventricular contractility is increased by 15.9 fold and for each increase of 1 unit of miR-101 in a patient body fluid the likelihood of classifying said patient into the high risk category of developing a reduced left ventricular contractility is decreased by 5.2 fold and for each increase of 1 unit of miR-150 in a patient body fluid, the likelihood of classifying said patient into the high risk category of developing a reduced left ventricular contractility is decreased by 12.1 fold and for an at least 3-fold increase of Nt-pro-BNP in a patient body fluid there is a high likelihood that a patient will develop a reduced left ventricular contractility.
  • kits for determining the prognosis of a patient diagnosed with an acute myocardial infarction preferably comprise devices and reagents for measuring the Nt-pro-BNP level in a patient sample, and devices and reagents for measuring the panel of 4 miRNAs of the invention and instructions for performing the assays.
  • the kits may contain one or more means for converting the Nt-pro-BNP levels and miRNA panel levels to a prognosis.
  • the application further discloses methods for the treatment of left ventricular modeling.
  • the treatment is conditional to the value of the prognostic score obtained through the method for predicting and/or monitoring the prognosis of a patient having suffered from an acute myocardial infarction of the present invention.
  • ventricular remodeling or reduced left ventricular cardiac contractility
  • methods useful for the treatment of left ventricular remodeling based on the supplementation of miR-150 and/or miR-101 and/or in combination with the inhibition of miR-16 and/or miR-27a.
  • the wording 'at least one short interfering nucleic acid capable of encoding a miRNA selected from the list consisting of miR-101 and miR-150' refers to the supplementation of the miRNA expression of miR-150 or miR-101 which is downregulated in patients predicted with a prognosis of developing cardiac left ventricular remodeling.
  • a short interfering nucleic acid capable of encoding a miRNA can for example be a microRNA, a short interfering RNA, a double-stranded RNA or a short hairpin RNA.
  • a short interfering nucleic acid of the present invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • chemically-modified short interfering nucleic acids improves various properties of native short interfering nucleic acid molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
  • Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency.
  • modifications base, sugar and/or phosphate
  • modifications base, sugar and/or phosphate modifications that prevent their degradation by serum ribonucleases can increase their potency.
  • sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups.
  • the short interfering nucleic acid molecule is double stranded and each strand of the short interfering nucleic acid molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
  • each strand of the short interfering nucleic acids comprises about 16 to about 25 nucleotides.
  • a short interfering nucleic acid sequence is substantially similar to the sequence of the selected miRNA, or is a short interfering nucleic acid sequence which is identical to the selected miRNA sequence at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases.
  • the short interfering nucleic acid sequence is a sequence that is substantially similar to the sequence of an miRNA, or is a short interfering nucleic acid sequence that is different than the miRNA sequence at all but up to one base.
  • a miRNA is supplemented by delivering an siRNA having a sequence that comprises the sequence, or a substantially similar sequence, of the miRNA.
  • miRNAs are supplemented by delivering miRNAs encoded by shRNA vectors. Such technologies for delivery exogenous microRNAs to cells are well known in the art.
  • a short interfering nucleic acid capable of inhibiting a miRNA refers to the inhibition of a selected miRNA function.
  • a miRNA in itself inhibits the function of the mRNAs it targets and, as a result, inhibits expression of the polypeptides encoded by the mRNAs.
  • blocking (partially or totally) or inhibiting the activity of a selected miRNA can effectively induce, or restore, expression of a polypeptide whose expression is inhibited (derepress the polypeptide).
  • derepression of polypeptides encoded by mRNA targets of a selected miRNA is accomplished by inhibiting the miRNA activity in cells through any one of a variety of methods.
  • blocking the activity of a miRNA can be accomplished by hybridization with a short interfering nucleic acid that is complementary, or substantially complementary to, the miRNA, thereby blocking interaction of the miRNA with its target mRNA.
  • a short interfering nucleic acid that is substantially complementary to a miRNA is a short interfering nucleic acid that is capable of hybridizing with a selected miRNA, thereby blocking the miRNA's activity.
  • a short interfering nucleic acid that is substantially complementary to a miRNA is a short interfering nucleic acid that is complementary with the miRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases.
  • a short interfering nucleic acid sequence is a sequence that is substantially complementary to a miRNA, or is a short interfering nucleic acid sequence that is complementary with the miRNA at, at least, one base.
  • antisense oligonucleotides including chemically modified antisense oligonucleotides - such as 2' O-methyl, locked nucleic acid (LNA) - inhibit miRNA activity by hybridization with guide strands of mature miRNAs, thereby blocking their interactions with target mRNAs.
  • 'antagomirs' are phosphorothioate modified oligonucleotides that can specifically block a selected miRNA in vivo (see for example Kurtzfeldt, J. et al. (2005) Nature 438, 685-689 ).
  • microRNA inhibitors termed miRNA sponges, can be expressed in cells from transgenes (see for example Ebert, M.S. (2007) Nature Methods, 12 ) . These miRNA sponges specifically inhibit selected miRNAs through a complementary heptameric seed sequence and even an entire family of miRNAs can be silenced using a single sponge sequence. Other methods for silencing miRNA function in cells will be apparent to one of ordinary skill in the art.
  • compositions as described herein before for the treatment of a human subject which may be a pediatric, an adult or a geriatric subject, wherein said human subject is predicted to develop left ventricular remodeling.
  • treatment, or treating includes amelioration, cure of a left ventricular remodeling.
  • the invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be various written materials (written information) such as instructions (indicia) for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • compositions of the present invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally.
  • the active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions of this invention are said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • suitable carriers are well-known in the art. It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present invention.
  • An effective amount, also referred to as a therapeutically effective amount, of a short interfering nucleic acid as described herein before is an amount sufficient to ameliorate at least one adverse effect associated with expression, or reduced expression, of the selected microRNA in a cell (for example a myocardial cell) or in an individual in need of such inhibition or supplementation.
  • the therapeutically effective amount of the short interfering nucleic acid (active agent) to be included in pharmaceutical compositions depends, in each case, upon several factors, e.g. the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc. Generally, an amount of active agent is included in each dosage form to provide from about 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg. One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount.
  • small interfering nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording, for example, the possibility of combination therapies with known drugs, or intermittent treatment with combinations of small interfering nucleic acids and/or other chemical or biological molecules).
  • therapeutic short interfering nucleic acids of the invention delivered exogenously are optimally stable within cells until translation of the target mRNA has been inhibited long enough to reduce the levels of the protein. This period of time varies between hours to days depending upon the disease state.
  • These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents.
  • the administration of the herein described small interfering nucleic acid molecules to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • the dosage of the administered nucleic acid molecule will vary depending upon such factors as the patient's age, weight, sex, general medical condition, and previous medical history. Typically, it is desirable to provide the recipient with a dosage of the molecule which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage may also be administered. In some embodiments, it may be desirable to target delivery of a therapeutic to the heart, while limiting delivery of the therapeutic to other organs. This may be accomplished by any one of a number of methods known in the art. The delivery to the heart of a pharmaceutical formulation described herein comprises coronary artery infusion.
  • the coronary artery infusion involves inserting a catheter through the femoral artery and passing the catheter through the aorta to the beginning of the coronary artery.
  • the targeted delivery of a therapeutic to the heart involves using antibody-protamine fusion proteins, such as those previously described ( Song E et al. (2005) Nature Biotechnology Vol. 23(6), 709-717 ) to deliver the small interfering nucleic acids disclosed herein. While it is possible for the agents to be administered as the raw substances, it is preferable, in view of their potency, to present them as a pharmaceutical formulation.
  • the formulations for human use comprise the agent, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof or deleterious to the inhibitory function of the active agent.
  • the formulations should not include oxidizing agents and other substances with which the agents are known to be incompatible.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the agent with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agent with the carrier(s) and then, if necessary, dividing the product into unit dosages thereof.
  • Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparations of the agents, which are preferably isotonic with the blood of the recipient.
  • suitable such carrier solutions include phosphate buffered saline, saline, water, lactated ringers or dextrose (5% in water).
  • Such formulations may be conveniently prepared by admixing the agent with water to produce a solution or suspension, which is filled into a sterile container and sealed against bacterial contamination.
  • sterile materials are used under aseptic manufacturing conditions to avoid the need for terminal sterilization.
  • Such formulations may optionally contain one or more additional ingredients among which may be mentioned preservatives, such as methyl hydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride.
  • preservatives such as methyl hydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride.
  • Buffers may also be included to provide a suitable pH value for the formulation. Suitable such materials include sodium phosphate and acetate. Sodium chloride or glycerin may be used to render a formulation isotonic with the blood.
  • the formulation may be filled into the containers under an inert atmosphere such as nitrogen or may contain an antioxidant, and are conveniently presented in unit dose or multidose form, for example, in a sealed ampoule.
  • Table 1 shows the demographic features of patients of the studied population. Among the 150 patients enrolled 79 patients evidenced preserved LV contractility at follow-up (WMIS ⁇ 1.2) and 71 patients had a loss of LV contractility. Comparisons between these 2 groups of patients revealed that patients with impaired contractility had higher levels of troponin I, creatine kinase and Nt-pro-BNP at discharge than patients with preserved contractility. Diuretics were more often prescribed in these patients, and they had a higher risk of developing chronic heart failure. Table 1.
  • ACE angiotensin-converting enzyme
  • BNP brain natriuretic peptide
  • CABG coronary artery bypass grafting
  • CHF congestive heart failure
  • CK creatine kinase
  • FH familial hypercholesterolemia
  • MI myocardial infarction
  • PCI percutaneous coronary intervention
  • STEMI ST-elevation myocardial infarction.
  • Table 2 shows the parameters of LV function as assessed by echocardiography, at discharge from the hospital and at 6-months follow-up. Patients who subsequently developed a loss of LV contractility had lower EF and higher LV volumes and diameters compared to patients with preserved LV contractility, at discharge from the hospital as well as after 6 months. Table 2.
  • LVEDV end-diastolic volume
  • LVESV end-systolic volume
  • LVEF ejection fraction
  • LVIDd end-diastolic diameter
  • LVIDs end-systolic diameter
  • LV contractility at follow-up was assessed by echocardiographic determination of WMIS. High WMIS indicates an impairment of LV contractility. 55 patients had a WMIS equal to 1, indicating a fully preserved contractility in these patients. Due to this left censoring of WMIS values at 1, censored regression (aka "Tobit regression") was used for prediction analysis. To compare the predictive value of miRNAs over classical markers, 2 multivariable models were built.
  • the first model included the following parameters: age, gender, smoking habit, diabetes, hypertension, hypercholesterolemia, antecedent of MI, infarct type (STEMI vs NSTEMI), infarct territory (anterior vs inferior), and Nt-pro-BNP level at discharge.
  • the second model included all the parameters of model 1 and the expression values of a panel of 4 miRNAs, miR-16/27a/101/150, measured in plasma samples obtained at discharge.
  • Figure 1A shows the odds ratios (OR) of each variable in model 1.
  • Infarct type, infarct territory and Nt-pro-BNP were significant predictors of WMIS. Patients with anterior STEMI and elevated Nt-pro-BNP were at higher risk of impaired contractility.
  • Figure 1B shows that miR-27a and miR-150 significantly contributed to the predictive value of model 2. The predictive values of miR-16 and miR-101 were of borderline significance.
  • the Wald chi square test indicates the significance of the model.
  • the likelihood ratio test compares the predictive value of a model with miRNAs to model 1.
  • AIC Akaike information criteria.
  • Bootstrap internal validation was used to test the strength of the models with combinations of miRNAs ( Figure 2 ).
  • the principle of this test is to calculate the predictive value of the model after resampling patient from the original sample.
  • the model including the 4 miRNAs was the best predictor in 29% of the 150 iterations performed.
  • MiR-27a was selected in 13% of cases and was included in all top models, showing its significant contribution to the prediction.
  • WMIS was used as a continuous variable.
  • WMIS value 1.2
  • WMIS ⁇ 1.2 the probability of belonging to the group of patients that will have impaired LV contractility
  • WMIS ⁇ 1.2 the probability of belonging to the group of patients that will not have impaired LV contractility
  • the logistic regression output can thereafter be used as a classifier by prescribing that a sample will be classified in the group of patient that will have impaired LV contractility if P is greater than 0.5, or 50%.
  • Predicting variables included the expression levels of the four miRNAs (in log-scale), the level of Nt-pro-BNP and the other clinical variables.
  • model 1 and model 2 2 multivariable models were built: model 3 includes all clinical variables and Nt-pro-BNP, and model 4 includes all variables of model 3 and the 4 miRNAs panel. Odds ratio (OR) for each variable in both models are shown in Figure 3 .
  • variable in the formula indicates the expression values of this particular miRNA in the patient as determined by quantitative RT-PCR as described in Material and Methods.
  • variable indicates the concentration of Nt-pro-BNP in the patient blood as determined by immune-assay as described in Material and Methods.
  • variables in the formula are binary, except for age which was considered as a continuous variable.
  • Odds ratio indicates the contribution of each variable to the prediction. OR below 1 indicates a negative association between a considered variable and the outcome of the patient; OR above 1 indicates a positive association between a considered variable and the outcome of the patient. OR were obtained with R version 2.13.1 with Hmisc, pROC, aod, lmtest and AER packages.
  • Each OR is associated with 95% CI and P value indicating the statistical significance of the variable in the model.
  • Each value of the odd ratio as mentioned in the formula can be replaced by any value within its corresponding 95% CI.
  • OR value can be from 0.01 to 0.48.
  • the intercept (In 8.51x10E-5) is a constant.
  • the Wald chi square test indicates the significance of the model.
  • the likelihood ratio test compares the predictive value of a model with miRNAs to model 1.
  • AIC Akaike information criteria.
  • the continuous version of the Net Reclassification Index and the Integrated Discrimination Improvement were computed to determine the ability of miRNAs to correctly reclassify patients misclassified by model 3 (Table 5). These are indexes of the change in classification of patients from one category of WMIS to another category ( ⁇ 1.2 or > 1.2).
  • Several combinations of miRNAs also provided significant reclassifications, such as miR-16/150, miR-27a/150, miR-16/27a/150, or miR-27a/101/150.
  • a main advantage of new biomarkers is to improve the classification of patients with intermediate phenotypes, which are difficult to classify using existing biomarkers.
  • 25 patients had moderate LV dysfunction (1.2 ⁇ WMIS ⁇ 1.4) and 24 had no LV dysfunction (1 ⁇ WMIS ⁇ 1.2).
  • Logistic regression and leave-one-out cross validation were used in these analyses. Two models were built, one with clinical variables and Nt-pro-BNP and one with clinical variables, Nt-pro-BNP and the 4 miRNA panel.
  • the model with clinical variables and Nt-pro-BNP had a specificity of 75%, but also a poor sensitivity of 48%.
  • the 4 miRNAs panel increased the sensitivity to 60%, while maintaining the specificity at 75%. With miRNAs, the positive predictive value was increased from 67% to 71%, and the negative predictive value was increased from 58% to 64%. Therefore, the 4 miRNAs panel improved the prognostication of patients with ambiguous phenotype, particularly dyskinetic patients.
  • ST-elevation AMI (STEMI) (Table 1) were enrolled in this study.
  • the diagnosis of AMI was based on presentation with appropriate symptoms of myocardial ischemia, dynamic ST segment elevation, and increase in markers of myocyte necrosis [creatine kinase (CK) and troponin I (TnI)] to above twice the upper limit of the normal range.
  • Venous blood samples were collected in EDTA-aprotinin tubes, immediately prior to discharge (day 3-4 after AMI). Samples were centrifuged within 30 minutes and plasma stored in aliquots at -80°C.
  • LV remodelling was assessed by echocardiography, as described 9 , conducted by a single operator (DK) at discharge and at a median of 176 days (range 138-262 days) after AMI.
  • LV contractility was evaluated by the LV wall motion index score (WMIS), using a standard 16-segment model from para-sternal long- and short-axis and apical two- and four-chamber views. Each LV segment was scored as 0, hyperkinetic; 1, normal; 2, hypokinetic; 3, akinetic; 4, dyskinetic.
  • Expression values were normalized using the mean Ct obtained from the spiked-in controls [calculation formula: 2 exp (mean Ct spiked-in controls - Ct target miRNA)] and log-transformed.
  • the detection limit of the PCR assay was -7.2, which is the log transformation of the minimum expression detected divided by 10.
  • N-terminal (amino acids 1 to 12) and C-terminal (amino acids 65 to 76) of the human Nt-pro-BNP were used to raise rabbit polyclonal antibodies.
  • IgG from the sera was purified on protein A sepharose columns.
  • the C-terminal-directed antibody (0.5 ⁇ g in 100 ⁇ L for each well) was immobilized onto ELISA plates.
  • the N-terminal antibody was affinity purified and biotinylated using biotin-X-N-hydroxysuccinimide ester (Calbiochem).
  • Prediction analyses were performed with R version 2.13.1 with Hmisc, pROC, aod, lmtest and AER packages. A P-value was considered significant when lower than 0.05. Clinical features were coded as 1 for presence and 0 for absence. Male was chosen as the reference level for sex in regression models. No data were missing thus no imputation method was performed.
  • Model parameter estimates were tested for nullity using a Z test in censored regression and a Wald Chi-square test in logistic regression. Residuals were analysed graphically both to detect nonlinear relationships between each variable in a model and WMIS, and to check normality assumptions for Tobit regression. For logistic regression, odd ratios (OR) and 95% confidence intervals (CI) were obtained by exponential transformation, and are shown in Figures 1 to 3 .
  • Bootstrap internal validation was used to correct all measures of model performance for optimisation. For each bootstrap sample (i.e. a random sample of individuals with the same size as the original sample where a given patient can appear several times), the whole model selection process was performed again to select the best model according to AIC criterion; the original sample was then tested with this model. In order to evaluate optimisation, NRI and IDI were computed with the test (i.e. original) set and subtracted to the same measures computed with the bootstrap sample. Afterwards optimisation was averaged across 150 bootstrap replications and finally subtracted to the measures obtained with the original sample as a training set.
  • the present invention refers to the following nucleotide and amino acid sequences:
  • the selection of the 4 miRNAs of the present invention was a long process, starting from an initial hypothesis that circulating miRNAs may be associated with remodelling post MI.
  • the first step was to perform microarray experiments in blood samples from 2 small groups of MI patients, one with, and one without, remodelling. From the 695 miRNAs represented on the microarrays, we isolated 271 miRNAs that were differentially expressed between patients with and without remodelling. The complete data are in Table S1. To isolate from these 271 those with potential link to remodelling, we used a systems-based approach with interaction networks. This permitted the identification of 10 miRNAs with the highest probability of association with remodelling. From these, we selected miR-27a/-101/-150 because of their high level of expression and differential expression between remodelers and non-remodelers.
  • miR-16/- 92a/486 are miR-16/- 92a/486, because of their high expression and differential expression in microarrays, and because they had been noted in Goretti et al., J. Leukoc. Biol. (2013 ).
  • MI myocardial infarction
  • MicroRNA-150 A novel marker of left ventricular remodeling after acute myocardial infarction. Circ. Cardiovasc Genet. 6:290-298 )
  • FIG. 5 shows systems-based identification of candidate miRNAs.
  • Fig. 5A Network of interactions between proteins known to be associated with LV remodelling in humans (dark grey nodes) and 26 interacting proteins (light grey). From the 13 proteins associated with LV remodelling, only 11 had known protein-protein interactions in at least 2 queried databases.
  • B Network of interactions between the 11 proteins associated with LV remodelling (dark grey), their 26 interactors light grey) and their 265 target miRNAs (medium grey). This network was built with CytoScape.
  • C Global view of the network containing 15 modules. This view was built with Polar Mapper.
  • D Discharge plasma levels of miR-27a/101/150 in 60 AMI patients of the derivation cohort, as measured by quantitative PCR.
  • Figure 6 shows expression of differentiation-related genes in early endothelial progenitor cells treated by anti-miR-16.
  • A_25_P00012 834 hsa-miR-652 1845.44 1593.59 1308.95 1307.84 - - - - A_25_P00010 459 hsa-miR-660 1553.98 1478.56 1371.66 1476.23 - - - - A_25_P00010 799 hsa-miR-663 10362.67 10492.30 13259.60 13393.78 - - - - A_25_P00010 800 hsa-miR-663 4224.00 3623.73 5824.41 5721.62 - - - - A_25_P00013 004 hsa-miR-665 1225.56 940.74 - - - - - - A_25_P00012 860 hsa-miR-671-5p 2936.63

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Claims (10)

  1. Kit zum Überwachen der Prognose eines Patienten, der an akuter Myokardischämie gelitten hat, bestehend aus (a) Sonden zum Messen einer Gruppe von miRNA-Biomarkern in einer Körperflüssigkeitsprobe des Patienten, wobei die Gruppe der miRNA-Biomarker besteht aus:
    (a) miR-16, das durch SEQ ID NO: 1 codiert wird,
    (b) miR-27a, das durch SEQ ID NO: 2 codiert wird,
    (c) miR-101, das durch SEQ ID NO: 3 codiert wird, und
    (d) miR-150, das durch SEQ ID NO: 4 codiert wird,
    und gegebenenfalls (b) Nachweisreagenzien zum Messen von Nt-pro-BNP wie in SEQ ID NO: 5 gezeigt, in einer Körperflüssigkeitsprobe des Patienten.
  2. Kit nach Anspruch 1, wobei die Sonden zum Messen der Gruppe von miRNAs zum Nachweis durch quantitative RT-PCR geeignet sind
    und die Nachweisreagenzien zum Messen des Nt-proBNP-Spiegels für den Nachweis durch Immunassay geeignet sind.
  3. Verwendung eines Kits nach Anspruch 1 oder 2 zum Überwachen der Prognose eines Patienten, der an akuter Myokardischämie gelitten hat.
  4. Verfahren zur Vorhersage und/oder Überwachung der Prognose des linksventrikulären Remodeling bei einem Patienten, wobei der Patient an einem akuten Myokardinfarkt gelitten hat, umfassend
    i) Bestimmen der Spiegel von miR-16, miR-27a, miR-101 und miR-150 in einer Körperflüssigkeitsprobe aus dem Patienten und
    ii) Korrelieren der miRNA-Spiegel mit:
    a) Spiegeln, die in einer Population von Kontrollpatienten beobachtet werden, die entweder nicht an einer AMI gelitten haben oder die an einer AMI gelitten haben und die linksventrikuläre Kontraktilität bewahrt haben, wobei ein statistisch signifikanter Anstieg der miR-16- und mi-R27a-Spiegel und eine statistisch signifikante Abnahme der miR-150- und miR-101-Spiegel im Vergleich zur Kontrolle eine beeinträchtigte linksventrikuläre Kontraktilität oder einen Verlauf zu beeinträchtigter linksventrikulärer Kontraktilität anzeigen und gegebenenfalls ebenfalls Bestimmen eines Anstiegs der Nt-pro-BNP-Spiegel durch Vergleich mit der Kontrolle, oder
    b) mit einem zuvor eingesetzten Klassifikationsmodell, wobei das Modell entwickelt wurde durch Anpassen von Daten aus einer Studie einer Population von Patienten, und die angepassten Daten Biomarkerspiegel umfassen und Umwandlung zur Entwicklung von linksventrikulärem Remodeling in der ausgewählten Population von Patienten und gegebenenfalls Korrelieren der Nt-pro-BNP-Spiegel mit dem Klassifikationsmodell, und
    iii) Gewinnen eines prognostischen Werts, der das Risiko für die Entwicklung eines linksventrikulären Remodeling angibt, indem die Chancen-Verhältnisse von nur miRNAs gegebenenfalls in Kombination mit den Nt-pro-BNP-Spiegeln eingesetzt werden.
  5. Verfahren nach Anspruch 4, wobei der Patient einen WMIS-Wert zwischen 1 und 1,4 hat.
  6. Verfahren zum Bewerten der Wirksamkeit einer Behandlung für einen Patienten, der an einem akuten Myokardinfarkt gelitten hat und eine Wahrscheinlichkeit hat, eine reduzierte linksventrikuläre Kontraktilität zu entwickeln, wobei das Verfahren umfasst: i) Bestimmen der Spiegel von miR-16, miR-27a, miR-101 und miR-150 in einer Körperflüssigkeitsprobe des Patienten, ii) Bestimmen des Nt-pro-BNP-Spiegels in einer Körperflüssigkeitsprobe aus dem Patienten, iii) Bestimmen der Spiegel von miR-16, miR-27a, miR-101 und miR-150 und des Nt-pro-BNP-Spiegels in einer Körperflüssigkeitsprobe aus dem Patienten nach der Behandlung, iv) Vergleichen der Ergebnisse nur von i) und ii) mit den Ergebnissen von iii), wobei ein Unterschied zwischen den Ergebnissen von i), ii) und iii) eine Wirkung der Behandlung anzeigt.
  7. Verfahren nach Anspruch 6, wobei der Patient einen WMIS-Wert zwischen 1 und 1,4 aufweist.
  8. Verfahren nach einem der Ansprüche 4 bis 7, wobei die Körperflüssigkeit Blut, Serum, Plasma, Cerebrospinalflüssigkeit, Speichel oder Urin, vorzugsweise Blut, Plasma oder Serum ist.
  9. Verfahren zum Einsetzen eines Klassifikationsmodells, umfassend
    i) Einsetzen der Spiegel einer Biomarkergruppe, bestehend aus miR-16, miR-27a, miR-101 und miR-150 in einer Körperflüssigkeitsprobe aus einer Population von MI-Patienten, und gegebenenfalls Einsetzen des Nt-pro-BNP-Spiegels und
    ii) Umwandeln der Spiegel dieser Biomarkergruppe und gegebenenfalls des Nt-pro-BNP-Spiegels zur Entwicklung von linksventrikulärem Remodeling in der ausgewählten Patientenpopulation,
    wobei eine Prognose für ein Risiko zur Entwicklung von linksventrikulärem Remodeling durch Einsetzen der Chancenverhältnisse der Biomarkergruppe von miRNAs und gegebenenfalls von Nt-pro-BNP erstellt wird.
  10. Verfahren nach Anspruch 9, wobei die Wahrscheinlichkeit P für die Entwicklung von linksventrikulären Remodeling berechnet wird durch: P = exp X / 1 + exp X ,
    Figure imgb0010
     wobei X = miR150 x ln 0,08 + miR101 x ln 0,19 + miR27a x ln 15,9 + miR16 x ln 4,18 + Nt-pro-BNP x ln 3,97 + Bereich x ln 2,29 + STEM/NSTEMI x ln 1,68 + vorheriger MI x ln 8,87 + Hypercholesterinämie x ln 1,63 + Hypertonie x ln 1,00 + Diabetes x ln 0,70 + Rauchgewohnheit x ln 1,49 + Geschlecht x ln 1,29 + Alter x ln 1,00 + ln 8,51x10E-5 und wobei, wenn P > 0,5 ist, dann ein signifikantes Risiko des Remodelings besteht (WMIS> 1,2) und wenn P <= 0,5 ist, dann ein geringes oder gar kein Risiko der Remodeling besteht (WMIS <= 1,2).
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